Screening for Resistance to Smut Disease of Twenty Seven Sugarcane Varieties ﻣﺳﺢ ﻣﻘﺎوﻣﺔ ﻣرض اﻟﺳوﯾد ﻓﻲ ﺳﺑﻌﺔ وﻋﺷرون ﺻﻧف ﻣن ﻗﺻب اﻟﺳﻛر

Sudan University of Science and Technology College of Graduate Studies

A thesis submitted in partial fulfillment of the requirements for the M.Sc. Degree in Plant Protection

By: Mohammed Ali Ahmed Hassin

Sudan University of Science and Technology

Department of Plant Protection

B.Sc. Agric. (Honors), 2012.

Supervisor: Dr. Ibrahim Saeed Mohamed

Department of Plant Protection

Shambat-College of Agricultural Studies

Sudan University of Science and Technology

2016 اﻵﯾﺔ

ﻗﺎل ﺗﻌﺎﱄ:

وَﻫُﻮ َ اﻟﱠﺬِي أَﻧـْﺰَلَ ﻣِﻦَ اﻟﺴﱠﻤﺎءِ (ﻣﺎءً ﻓَﺄَﺧْﺮَﺟْ ﻨﺎ ﺑِﻪِ ﻧَﺒﺎتَ ﻛُﻞﱢ ﺷَﻲ ْ ءٍ ﻓَﺄَﺧْﺮَﺟْ ﻨﺎ ﻣِﻨْﻪُ ﺧَﻀِﺮً ا ﳔُْﺮِجُ ﻣِﻨْﻪُ ﺣَ ﺒﺎ ﻣُﱰَ اﻛِﺒًﺎ وَﻣِﻦَ اﻟﻨﱠﺨْﻞِ ﻣِﻦْ ﻃَﻠْﻌِﻬﺎ ﻗِﻨْﻮانٌ داﻧِﻴ َ ﺔٌ وَﺟَﻨﱠﺎتٍ ﻣِﻦْ أَﻋْﻨﺎبٍ وَ اﻟﺰﱠﻳـْﺘُﻮنَ وَ اﻟﺮﱡﻣﱠﺎنَ ﻣُﺸْﺘَﺒِﻬً ﺎ وَﻏَﻴـْﺮَ ﻣُﺘَﺸﺎﺑِﻪٍ اﻧْﻈُﺮُوا إِﱃ ﲦََﺮِﻩِ إِذا أَﲦَْﺮ َ وَ ﻳـَ ﻨْﻌِﻪِ إِنﱠ ﰲِ ذﻟِﻜُﻢ ْ ﻵَ ﻳﺎتٍ ﻟِﻘَﻮ ْ مٍ ﻳـُﺆْﻣِ ﻨُﻮنَ ((99))

ﺻﺪق اﷲ اﻟﻌﻈﻴﻢ

ﺳﻮرة اﻷﻧﻌﺎم((99))

Dedication To my mother

To my father

To my sisters and brothers

To my family

To my teachers

To my colleagues and friends

With love and respect.

Mohammed

ACKNOWLEDGMENTS

All thanks are due to Almighty Allah (SWT) who gave me health and strength, and helped me tremendously to produce this work.

I would like to express my thanks to my supervisor Dr. Ibrahim Saeed Mohamed College of Agricultural studies, Sudan University of science and technology, who devoted most of his time to teach us on various disciplines of scientific research.

Thanks are also extended to my dear teachers Khalid Bukhari for their continuous and unlimited helps during this study. From whose blessed hand the statistical analyses were produced.

Also Special Thanks are due to my colleagues at the M.Sc. who helped me during the experiments. Grateful thanks are due to all the staff of the Department of Plant Protection, College of Agricultural Studies, Sudan University of Science and Technology.

III

List of Contents

Title Page No. I...... اﻵﯾﺔ Dedication ...... II ACKNOWLEDGMENTS ...... III List of Contents ...... IV List of Tables ...... VI List of Figures ...... VII List of Plates ...... VIII Abstract ...... IX X ...... ﻣﻠﺨﺺ اﻟﺒﺤﺚ CHAPTER ONE ...... 1 INTRODUCTION ...... 1 CHAPTER TWO ...... 4 LITREATURE REVIEW ...... 4 2.1. Sugarcane (Saccharum spp.) ...... 4 2.1.1 Description of the crop ...... 4 2.1.2 Sugarcane production worldwide ...... 6 2.1.3 Yield and quality ...... 7 2.1.4 Nutritional value of Sugarcane: ...... 7 2.2 Sugar cane production in Sudan ...... 7 2.2.1 Constraints of sugar cane production ...... 8 2.2 Sugarcane smut disease ...... 8 2.3.1 Classification of sugarcane smut pathogen {Sporisorium scitamineum (Syd.) M. Piepenbr; Stoll, M. & Oberw, 2002} ...... 10 2.3.2Symptoms ...... 11 2.3.3 The pathogen: ...... 12 2.3.4 Variability ...... 13 2.3.5 Disease cycle ...... 16 IV

2.3.6 Dissemination of spores ...... 17 2.3.7 Environment ...... 18 2.3.8 Economic Impact ...... 18 2.3.9 Prevention and Control ...... 19 CHAPTER THREE ...... 21 MATERIALS AND METHODS ...... 21 3.1. Location of study: ...... 21 3.2. Test varieties ...... 21 3.3. Collection and maintenance of S. scitamineum inoculum ...... 21 3.4. Seedbed preparation and planting materials ...... 22 3.5. Preparation of sugarcane differentials: ...... 22 3.6. Inoculation protocol/ methods and field design: ...... 22 3.6.1. Dip methods (DM): ...... 22 3.6.2 Natural spreading method (NSM): ...... 23 3.7. Disease incidence and assessment of resistance ...... 23 3.8. Data collection ...... 23 3.9. Statistical analysis ...... 24 3.10. Evaluation of sugarcane germplasm against smut: ...... 24 CHAPTER FOUR ...... 25 RESULTS ...... 25 4.1 Reaction of varieties inoculated with natural infection method ...... 25 4.2 Reaction of varieties to smut inoculated with dip inoculation method ... 29 CHAPTER FIVE ...... 35 DISCUSSION ...... 35 Conclusions and Recommendation ...... 37 Conclusions ...... 37 Recommendation ...... 38 References ...... 39 Appendices...... 46

List of Tables

Title Page No. Table 1: Numerical rating system for sugarcane smut according to (Satya Vir Beniwal, 1978)*...... 24 Table 2: Rating varieties of smut disease reactions to smut inoculated with natural infection method in plant cane (PC) and in first ratoon (R1)...... 26 Table 3: Rating varieties of smut disease reactions to smut inoculated with dipping infection method in plant cane PC and in first ratoon R1 ... 30 Table 4: Percentage of stool infection showed the correlation between the natural infection methods and dipping infection methods in plant cane. . 33

List of Figures

Title Page No. Figure 1: Percentage of smut stool infection in plant cane (PC) of sugarcane varieties inoculated with natural infection method ...... 27 Figure 2: Percentage of smut stool infection in first ratoon (R1) of sugarcane varieties inoculated with natural infection methods ...... 28 Figure 3: Percentage of smut stool infection in plant cane (PC) of sugarcane varieties inoculated with dip infection methods...... 31 Figure 4: Percentage of smut stool infection in fist ratoon (R1) of sugarcane varieties inoculated with dip infection methods ...... 32 Figure 5: Percentage of smut stool infection in plant cane (PC) of sugarcane varieties inoculated with natural infection methods ...... 34 Figure 6: Percentage of smut stool infection in plant cane (PC) of sugarcane varieties inoculated with dip infection methods ...... 34

VII

List of Plates

Title Page No.

Plate 1: Sugarcane smut whips on sugarcane cultivar CP88-1762 Credits: Philippe Rott, UF/IFAS...... 12

VIII

Abstract

Sugarcane smut (Sporisorium scitamineum) is considered cosmopolitan in distribution, and has been important in nearly every sugarcane producing country of the world and in Sudan as well. The control of the disease depends mainly on resistance of cane variety to the pathogen. In the Sudan smut draws greatest attention as the disease is prevalent on all sugar estates and at same times it seriously threatened the industry resulting in that two of the highest yielding varieties for both cane tonnage and sugar (Nco 310 and Nco 376) have been phased out of commercial cultivation. Since then the search for an effective control measures was emphasised. pressent study was aimed of the carried out at the research field of the Guneid Research Centre in seasons 2014-2015 screen newly introduced sugarcane varieties for thier resistance to smut disease using two methods of inoculation (natural and dipping inoculation). The results revealed that the test varieties reacted differently to inoculation with smut disease and hence they gave significantly variable level of infection. The percentage of stool infection ranged from 0% in variety CP 99-1894 that rated as highly resistant (HR) and 76% in variety BSR97051 which was rated as highly susceptible (HS). Moreover, out of the 27 varieties tested, five ones were reacted as highly resistant (HR) in plant cane, namely, (CP 99-1894, FG 03204, B 89640, DB 66113, DP 71060) and one variety FG 04754 rated 2R compared to check variety Co 6806 which is known as highly resistant (HR). These five varieties maintained similar reactions to smut in first ratoon by the two methods. The remaining varieties which reacted and rated less than resistant (R), their reaction rated from moderately to highly susceptible. The smut infection in these varieties was higher in first ratoon than in plant cane. This study proved categorically, that there is positive correlation in the results of the two methods used which suggests that the dip method is suitable for future studies. The study also recommended studies in disease epidemiology.

ﻣﻠﺨﺺ اﻟﺒﺤﺚ

ﯾﻌﺗﺑر ﻣرض اﻟﺗﻔﺣم ﻓﻲ ﻗﺻب اﻟﺳﻛر ﻣﻧﺗﺷر ﻋﺎﻟﻣﯾﺎ وذو أﻫﻣﯾﻪ ﻓﻲ ﻛل ﻣن اﻟدول اﻟﻣﻧﺗﺟﺔ ﻟﻘﺻب اﻟﺳﻛر ﻓﻲ اﻟﻌﺎﻟم وﻓﻰ اﻟﺳودان أﯾﺿﺎ. ﺗﻌﺗﻣد ﻣﻛﺎﻓﺣﺔ اﻟﻣرض ﺑﺻورة رﺋﯾﺳﯾﺔ ﻋﻠﻰ ﻣﻘﺎوﻣﺔ اﻟﻘﺻب ﻟﻠﻛﺎﺋن اﻟﻣﻣرض. ﻓﻲ اﻟﺳودان ﺣظﻲ ﻣرض اﻟﺳوﯾد ﺑﺎﻫﺗﻣﺎم ﻛﺑﯾر ﻻن اﻟﻣرض ﻣﺗواﺟد ﻓﻲ ﻛل ﻣﺷﺎرﯾﻊ اﻟﺳﻛر وﻓﻰ وﻗت ﻣﺎ ﻫدد اﻟﺻﻧﺎﻋﺔ ﺑﺷﻛل ﺧطﯾر ﻣﻣﺎ ﻧﺟم ﻋﻧﻪ ﺧروج أﻋﻠﻰ ﺻﻧﻔﯾن إﻧﺗﺎﺟﯾﺔ ﻣن ﺣﯾث اﻟﻘﺻب واﻟﺳﻛر و ﻫﻣﺎ NCo 310 and NCo 376 ﻣن اﻹﻧﺗﺎج اﻟﺗﺟﺎري. وﻣﻧذ ذﻟك اﻟﺣﯾن ﺗم اﻟﺗرﻛﯾز ﻋﻠﻰ اﻟﺑﺣث ﻋن طرق ﻓﻌﺎﻟﺔ ﻟﻠﻣﻛﺎﻓﺣﺔ. ﻫدﻓت اﻟدراﺳﺔ اﻟﺣﺎﻟﯾﺔ واﻟﺗﻲ ﻧﻔذت ﺑﻣزرﻋﺔ ﺑﺣوث ﻣرﻛز أﺑﺣﺎث اﻟﺟﻧﯾدﻓﻰ ﺳﻧﺔ 2014-2015 إﻟﻰ ﻓﺣص ﻣﻘﺎوﻣﺔ أﺻﻧﺎف ﻣن ﻗﺻب اﻟﺳﻛر اﺳﺗﺟﻠﺑت ﺣدﯾﺛﺎ ﺑﺎﺳﺗﻌﻣﺎل اﺛﻧﯾن ﻣن طرق اﻟﺗﻠﻘﯾﺢ (اﻟﺗﻠﻘﯾﺢ اﻟطﺑﯾﻌﻲ واﻟﻐﻣر). أظﻬرت اﻟﻧﺗﺎﺋﺞ أن رد ﻓﻌل اﻷﺻﻧﺎف اﻟﻣﺧﺗﺑرة ﺗﺟﺎﻩ اﻟﺗﻠﻘﯾﺢ ﺑﻣرض اﻟﺳوﯾد ﻛﺎن ﻣﺧﺗﻠﻔﺎ وﺑﺎﻟﺗﺎﻟﻲ أﻋطت ﻣﺳﺗوﯾﺎت إﺻﺎﺑﺔ ﻣﺧﺗﻠﻔﺔ ﻣﻌﻧوﯾﺎ. ﺗراوﺣت ﻧﺳﺑﺔ اﻹﺻﺎﺑﺔ ﻣﺎ ﺑﯾن 0% ﻓﻲ اﻟﺻﻧف CP 99-1894 واﻟﺗﻲ ﻗدرت أﻧﻬﺎ ذات ﻣﻘﺎوﻣﺔ ﻋﺎﻟﯾﺔ و %76ﻓﻲ اﻟﺻﻧف BSR97051 واﻟﺗﻲ ﻗدرت أﻧﻬﺎ ذات ﻗﺎﺑﻠﯾﺔ ﻟﻺﺻﺎﺑﺔ ﻋﺎﻟﯾﺔ. أﯾﺿﺎ ﻣن ﺑﯾن اﻟـ 27 ﺻﻧف اﻟﺗﻲ ﺗم إﺧﺗﺑﺎرﻫﺎ، ﺧﻣﺳﺔ أﺻﻧﺎف ﻛﺎﻧت رد ﻓﻌﻠﻬﺎ ﻋﺎﻟﯾﺔ اﻟﻣﻘﺎوﻣﺔ(HR) ﺑﺎﻟﺗﺣدﯾد ﻫم (CP 99-1894, FG 03204, B 89640, DB 66113, DP 71060) وﺻﻧف واﺣد FG 04754 ﺗم ﺗﻘدﯾرﻩ 2R ﻣﻘﺎرﻧﺔ ﺑﺎﻟﺻﻧف اﻟﺷﺎﻫد 6806 واﻟﻣﻌروف ﺑﻣﻘﺎوﻣﺔ اﻟﻌﺎﻟﯾﺔ (HR). ﻫذﻩ اﻷﺻﻧﺎف اﻟﺧﻣﺳﺔ ﺣﺎﻓظت ﻋﻠﻰ رد ﻓﻌل ﺿد اﻟﺳوﯾد ﻣﺷﺎﺑﻪ ﻓﻲ اﻟﺧﻠﻔﺔ اﻷوﻟﻰ (R1) . ﺑﻘﯾﺔ اﻷﺻﻧﺎف ﻛﺎﻧت اﻗل ﻣﻘﺎوﻣﺔ، اذ أن ﺗﻔﺎﻋﻠﻬﻣﺎ ﻗدر ﻣﺎ ﺑﯾن ﻣﺗوﺳط إﻟﻰ ﺷدﯾد اﻹﺻﺎﺑﺔ. أﻹﺻﺎﺑﺔ ﺑﺎﻟﺳوﯾد ﻓﻲ ﻫذﻩ اﻷﺻﻧﺎف ﻛﺎﻧت أﻋﻠﻰ ﻓﻲ اﻟﺧﻠﻔﺔ اﻷوﻟﻰ ﻣن اﻟﻘﺻب اﻟﻐرس. ﻫذﻩ اﻟدراﺳﺔ أﺛﺑﺗت ﺑﺻورة ﻣطﻠﻘﺔ أن ﻫﻧﺎﻟك إرﺗﺑﺎط ﻣوﺟب ﻓﻲ ﻧﺗﺎﺋﺞ اﻟطرﯾﻘﺗﯾن اﻟﻠذﯾن اﺳﺗﻌﻣﻠﺗﺎ اﻷﻣر اﻟذي ﯾﺷﯾر إﻟﻰ ﻣﻼﺋﻣﺔ طرﯾﻘﺔ اﻟﻐﻣر ﻓﻲ اﻟدراﺳﺎت اﻟﻣﺳﺗﻘﺑﻠﯾﺔ. اﻟدراﺳﺔ أﯾﺿﺎ أوﺻت ﺑدراﺳﺎت ﻓﻲ وﺑﺎﺋﯾﺔ اﻟﻣرض.

CHAPTER ONE

INTRODUCTION

Sugarcane is one of the several species of tall perennial true grasses of the genus Saccharum, family Poaceae, native to the warm temperate to tropical regions of South Asia, and used for sugar production (Peter, 1998). Different species likely originated in different locations, with Saccharum barberi originating in India and S. edule and S. officinarum in New Guinea. Approximately 70% of the sugar produced globally comes from open pollinated and hybrids S. officinarum (Peter, 2000).

The crop is the world's largest crop produced. FAO (2015) estimated that sugarcane was cultivated on about 26.0 million hectares, in more than 90 countries, with a worldwide harvest of 1.83 billion tons. Brazil is the largest producer of sugar cane in the world. The next five major producers, in decreasing amounts of production, were India, China, Thailand, Pakistan and Mexico (Draycott, 2006).

In Sudan, sugarcane was first grown in about 29 hectares in Berber area. Trails to grow sugarcane were conducted in late 1940s in Zandi area, southern Sudan (Bacon, 1952). Commercial production of sugarcane as industrial cash crop started in 1962/63 with the establishment of Guneid sugar factory. The industry was then rapidly expanded to include New Halfa sugar factory 1964/65, West Sennar sugar factory 1976/77, Assalaya sugar factory1979/81, Kenana sugar company 1980/81 and white Nile sugar company 2011/2012 the total acreage under sugar production is 376.5 thousand acres. The earliest sugarcane cultivars used were NCo310, NCo376 and Co527 which were replaced later on by the present high yielding ones (Co 775, Co 997 and Co 6806) (Obeid, 2005).

Numerous pathogens infect sugarcane, such as sugarcane grassy shoot disease caused by Phytoplasma, sugarcane smut caused by Sporisorium scitamineum, pokkah boeng caused by Fusarium moniliforme, gumming disease Xanthomonas axonopodis, and red rot disease caused by Colletotrichum falcatum. Viral diseases affecting sugarcane include sugarcane mosaic virus, maize streak virus, and sugarcane yellow leaf virus. The major constraint facing the productivity of sugarcane crop worldwide is sugarcane smut. It is one of the most serious diseases of this crop. Affected cane is severely stunted and production losses of 30-100% are common in susceptible varieties (Ferreira and Comstock, 1989; Croft et al 2000 and Solomon et al., 2000).

In Sudan, smut disease was first reported in the Sudan at Guneid on variety NCo 310 (Abu Gideiri, 1965; Nasr and Ahmed, 1974). Currently, it occurs in all sugar estates and causes considerable losses in susceptible varieties. Nasr and Ahmed (1974) reported 100% incidence thus led to the withdrawal of several excellent varieties i.e. NCO 310, Co 527 etc. from production. The use of the resistant sugarcane varieties such as Co 997 and Co 6806 has maintained the disease under some good control. However; occasional infections by S. scitamineum especially on Co 6806 (the number one variety) has become common probably suggesting resistance deterioration or variation in the pathogen population Marchelo,et al(2008)Since planting of resistance varieties is of paramount importance for disease control the current study aimed for screening of sugarcane varieties for resistance to smut disease with the following objectives:-

 To evaluate the resistance of some newly introduced sugarcane genotypes to sugarcane smut disease caused by Sporisorium scitamineum under field condition.  To compare different methods of screening of sugarcane genotypes for resistance to smut.

CHAPTER TWO

LITREATURE REVIEW

2.1. Sugarcane (Saccharum spp.)

Sugarcane is a robust, tall growing, perennial thick-stemmed and monocotyledonous grass crop cultivated in the tropical and subtropical regions of the world primarily for its ability to store high concentrations of sucrose or sugar in the stem (Rena, 1997). Sugarcane is a C4 plant having high efficiency in storing solar energy and most efficient converter of solar energy to sucrose. Sugarcane has essentially four growth phases: 1.Germination phase, 2. Tillering phase, 3. Grand growth phase, 4. Maturity and ripening phase. The crop is a long duration clonally propagated plant. In the right climate, the cane will grow in 12-14 months (plant cane) and, when cut, will re- grow in another 12 months (ratoon crop). The typical sugar content of mature cane would be 10% by weight but, usually, this varies depending on variety, location and even the season (Peter, 2000). The genus Saccharum has five important species viz., Saccharum officinarum, S. Sinense, barberi, S. robustum and S. spontaneum. The first three species are the cultivated species and the last two are wild ones. S. officinarum species is widely cultivated worldwide because of high sucrose content. 2.1.1 Description of the crop

Sugarcane is a tropical, perennial grass that forms lateral shoots at the base to produce multiple stems, typically three to four meters high and about five cm in diameter. The stems grow into cane stalk, which when mature constitutes approximately 75% of the entire plant. A mature stalk is typically composed of 11–16% fiber, 12–16% soluble sugars, 2–3%

4 non-sugars, and 63–73% water. The crop is sensitive to the climate, soil type, irrigation, fertilizers, insects, diseases and the harvest period. The plant is composed of four principal parts, root system, stalk, leaves and inflorescence. Sheath is green with red blotches; moderate to heavy bloom; scarious border prominent; sheath splitting occasional clasping; spines present on the middle of the sheath; deciduous. blade joint or transverse mark is purplish green; medium: fair bloom. Ligules’: medium; crescent form; symmetrical; gradually tapering towards the edges. The growth pattern includes germination; tillering and the underground nodes of primary shoots give rise to secondary shoots which in turn give rise to tertiary ones. As internodes extension becomes marked, apical dominance exercised by the leading shoots discourages further tiller production (Schueneman, 2002). Initially, tillering is profuse but is normally followed by a wave of mortality as soon as the rows close in. Miller and Lentine., (2002) reported that tillering increases with increasing light intensity and duration and more than 50 % of the number of the initial stalks die, mainly, due to light competition. They pointed out that the most important external factors influencing tillering are light, temperature, nutrition, moisture and spacing. The grand growth phase is also known as the cane formation phase in which upper internodes are rapidly formed and the lower internodes extend length and diameter. This phase is accompanied by the onset of the maturity phase which starts in the lower internodes of the stalk and proceeds gradually towards the upper ones. The maturing /harvesting age of sugarcane varies in the world ranges between 10 to 24 month depending on climatic condition (Alam, and Khan, 2001). Moreover, sugarcane is a perennial crop. Cane growth produced from the first year of cultivation is called plant cane whereas growth in subsequent year is called “ratoons” or “stubbles” The inflorescence of sugarcane 5 generally called 'arrow' which is an open panicle. It is long 30 centimeter or more and tapering. Cane is Medium-thick; slightly staggered; slightly oval in cross section, internal tissue yellow with purple tinge: rind hard; pith present as small cavity. Node and buds are slightly depressed; leaf scar slightly inclined. Buds are medium, plumpy, and ovate; occasionally hairs at the tip of the bud; inserted at leaf scar (Draycott, 2006).The sugarcane plant is a largely cross pollinated species with a low frequency of selfing and pollen is dispersed by wind and no insect vectors for sugarcane pollen are known (Mclntyre and Jackson, 2001).Pollen viability is a low under natural conditions and has a life of only 12 minutes and no viability after 35 minutes (More and Nuss, 1987). Modern sugarcane varieties that are commercially cultivated for sugar production are inter-specific hybrids between Saccharum spontaneum and Saccharum officinarum; and, are therefore generally referred to as Saccharum spp. (Anon, 1984; Cuadrado et al., 2004). 2.1.2 Sugarcane production worldwide

Three quarters of the world’s sugar is made from sugar cane in tropical zones located in the southern hemisphere. Leading sugarcane producers are Brazil, India, China, Thailand, Pakistan, and Mexico (FAO, 2015).The remainder is processed from sugar beets grown in temperate zones of the northern hemisphere. France, Germany, U.S., Russia, Ukraine and Turkey produce it from sugar beets. Currently, 70% of the world’s sugar is consumed in the country where are harvested; only 30% is traded outside country of origin. Global sugar consumption rises by about 2% per year, and has increased by 17%, from 128 million tons in the year 2000 to 150 million in 2006. The highest sugar consumption per capita is found in Brazil (59kg of sugar/annum), Mexico (53 kg sugar/annum) and Australia (50 kg of sugar /annum) (Workman, 2007).

2.1.3 Yield and quality

Yield and quality of sugarcane is a broad term making up the ultimate sugar yield per unit area. The cane yield is the outcome of the number of stalks per unit area together with the average stalk height and thickness. Mature cane, generally, consists of around 70% moisture and 30% dry matter. The dry matter consists of variable amounts of brix and fiber. The brix is the total soluble solids, which include mainly sugar (Pol) together with some salts and other organic non- sugar (Workman, 2007) 2.1.4 Nutritional value of Sugarcane:

The juice sugarcane per serving 28.35 grams contain energy-111.13 kJ (26.56 kcal), Carbohydrates-27.51 g, Protein-0.27 g, Calcium11.23 mg (1%), Iron 0.37 mg (3%), Potassium41.96 mg (1%), Sodium17.01 mg (1%) (Workman, 2007) 2.2 Sugar cane production in Sudan

In Sudan, sugarcane was first grown in about 29 hectares in Berber area. Trials to grow sugarcane were conducted in late 1940s in Zandi area, southern Sudan (Bacon, 1952). Commercial production of sugarcane as industrial cash crop started in 1962/63 with the establishment of Guneid sugar factory. The growing industry was then rapidly expanded to include New Halfa sugar factory 1964/65, West Sennar sugar factory 1976/77, Assalaya sugar factory 1979/81, Kenana sugar company 1980/81 and white Nile sugar company 2011/2012 the total acreage under sugar production 376.5 thousand acres. Farm were all irrigated and located in the central clay plain of Sudan. The projected annual output of these six schemes was 1195 thousand tons sugar (Obeid,. 2005). Sugarcane production in Sudan was supported by establishment of two research stations viz., Guneid sugarcane research station and Kenana sugar

7 company research stations. Sugarcane is grown under condition of high temperature, frequent irrigation, wide inter-row spacing (1.50-1.55m) and the crop receives substantial amount of fertilizers. The earliest sugarcane cultivars used were NCo310, NCo376 and Co527 which were replaced later on by the present high yielding main cultivars (Co775, Co797 and Co6806). Plant crop constituted generally 15% of the total cultivated area leaving the majority for ratoon crop (5-6 ratoons). The growth period of the crop is 14 month for plant cane and 11-12 month for rations. The crop is harvested using both manual and mechanical methods. Harvesting is normally done in winter months. Research finding coupled with management experience resulted in high improvement in cane varieties and cultural practices. These improvements, lately, boosted the average cane yield to reach 116 ton/ha in some sugarcane estates which is comparable to the international yield levels. 2.2.1 Constraints of sugar cane production

Sugar yield per unit area increases with optimum husbandry inputs, fertilizer, irrigation, weed control etc; However, it could adversely be affected by several other factors such as untimely planting, unsuitable harvest age and unfavorable climate. In practice, the planting period of sugar cane extends over many months with variable climatic conditions. Similarly, the harvest period extends over the variable weather conditions in the winter season. Lack of optimum planting times and harvest age in these periods constitutes major constraints for sugar cane production. 2.2 Sugarcane smut disease

Numerous pathogens infect sugarcane; bacterial, fungal, viral and nematodes diseases were reported worldwide. Among fungal diseases smut is the most devastating and threatening disease in all sugarcane growing areas World wide. 8

Sugarcane smut disease caused by the fungus Sporisorium scitamineum which was first reported from South Africa in 1877 on Chinese cane (Saccharum sinense); Thereafter, many observations were made in Africa, and Asia, in the following decades (Antoine, 1961; Presley, 1978). Smut remained confined to the Eastern hemisphere until 1940 when it was found in Argentina. It has since been recorded in most sugar cane producing countries of the world (Presley, 1978). Only, sugar industries of Eastern Australia, Fiji and Papua New Guinea are still free of the disease. Smut of sugarcane is reported to be a serious disease in most sugarcane producing countries and cane cause considerable yield losses and reduction in cane quality (Ferreira and Comstock, 1989). Croft et al (2000) reported yield losses between 20 – 100% depending on the cane variety. Some workers put losses at between 60 -70% (Fawcett, 1942; Raga et al., 1972; Alexander, 1995; Solomon et al., 2000). Smut was first reported in the Sudan at Guneid (on variety NCo 310 in Wad-Surur minor) in 1964/65 Abu Gideiri, (1965); Nasr and Ahmed, (1974). Thereafter it was subsequently, confirmed on 35 of the 46 varieties in the variety collections unit at that time, with an incidence of 30-40%. Currently it occurs in all sugar estates and causes considerable losses in susceptible varieties. Nasr and Ahmed (1974) reported 100% incidence in Sudan. It has also led to the withdrawal of several excellent varieties i.e. NCO 310, Co 527 etc. from production. The most likely source of this initial infection was never determined, but could have come with imported sugarcane material or wind-blown spores from neighboring countries. The use of the resistant sugarcane varieties such as Co 997 and Co 6806 has maintained the disease under some good control. However, occasional infections by S. scitamineum especially on Co 6806 (the number one variety) has become common probably suggesting variety deterioration (resistance erosion) or variation in the pathogen population. 9

Since then, successful disease management strategies and control require an understanding of the level of diversity in the pathogen population. Information on the genetic variability of the smut pathogen present in Sudan is of paramount important as this will enable plant breeders and pathologists to adopt/select appropriate breeding and control strategies including germplasm selection for increased resistance both in the introduced and current sugarcane genotypes in the country. The disease is sometimes referred to as “culmicolous” smut of sugarcane because it affects the stalk of the cane. At one time or another, sugarcane smut has been important in nearly every sugarcane growing country in the world. Sugarcane smut does not always pose a serious problem where it occurs. However, the disease may remain unnoticed for years, and then quickly devastate large areas of susceptible varieties. Hence, the disease has been called the “dread disease of sugarcane” by some and a “trivial disease with exaggerated yield losses” by others. Smut can cause significant tonnage losses as well as juice quality losses. Disease development is dependent on the environmental conditions and the resistance of the sugarcane cultivars (Royal Botanical Gardens, 2004).

2.3.1 Classification of sugarcane smut pathogen {Sporisorium scitamineum (Syd.) M. Piepenbr; Stoll, M. & Oberw, 2002}

Scientific classification

Kingdom: Fungi

Division: Basidiomycota

Class: Ustilaginomycetes

Order: Ustilaginales

Genus: Sporisorium Species: S. scitamineum Binomial name Sporisorium scitamineum {(Syd.) M. Piepenbr., M.Stoll& Oberw. 2002} Synonyms Ustilago scitaminea 10

2.3.2Symptoms

The most recognizable diagnostic feature of a smut infected plant is the emergence of a “smut whip” (Comstock, 2000). A “smut whip” is a curved, pencil-thick growth, gray to black in color that emerges from the top of the affected sugarcane plant (Figure, 1). These “whips” arise from the terminal bud or from lateral shoots on infected stalks. They can vary in length from a few inches to several feet long. The whip is composed partly of host plant tissue and partly of fungal tissue. Whips begin emerging from infected cane by 2–4 months of age with peak whip growth occurring at the sixth or seventh month.

Other smut symptoms may be evident before the characteristic whip is seen. Spindle leaves are erect before the whip emerges. Affected sugarcane plants may tiller profusely with the shoots being more spindly and erect with small narrow leaves (i.e., the cane appears “grass-like”). Less common symptoms are bud proliferation and leaf and stem galls. Recent modern techniques, molecular and serological based were used to diagnose the disease in early states. Technological advances in PCR- based methods, such as real-time PCR, allow fast, accurate detection and quantification of plant pathogens and are now being applied to practical problems. Singh et al., (2004) demonstrated that PCR assay was extremely sensitive in detecting the presence of the pathogen and yielded a positive response in plantlets inoculated with sporidia and observed that PCR assay was significantly better for smut detection than microscopy (Comstock, 2000). Dry and hot spring weather favors the disease. Plants grown under stress conditions are more prone to develop smut. In 1997, smut was observed on a resistant cultivar CP70-1133 which was grown on sand land under stressed conditions. Low incidences (less than 5%) of smut have been 11 observed in a few sand land fields of cultivars CP78-1628 and CP73- 1547 under stressed conditions. In 2014, several smut affected plants of cultivar CP88-1762 grown on muck soil were seen in an experimental field at the UF/IFAS Everglades Research and Education Center in Belle Glade, Florida (Figure 1).

Plate 1: Sugarcane smut whips on sugarcane cultivar CP88-1762 Credits: Philippe Rott, UF/IFAS

2.3.3 The pathogen:

Although occurrence of several races of S. scitamineum, the sugarcane smut fungus, has been reported, the race picture is poorly defined at this time. Part of the problem is due to sugarcane variety-environment interactions causing test-to-test variability regarding pathogenicity. Breakdown of resistance to smut due to appearance of new rust races has been confirmed in locations such as Hawaii and Taiwan, and worldwide genetic variation possibly linked to various pathotypes of S. scitamineum has also been reported (Sundar et al., 2012).

2.3.4 Variability

Information on the prevalence and distribution pattern of races/pathotype in S. scitamineum in an area is required for effective deployment of host resistance. Schenck (2003) recorded incidence of smut in one variety (H78-7750), considered to be completely resistant in several seed fields on Maui, indicating the possible emergence of a new race of the smut fungus in Hawaii. The new smut race was included in breeding program susceptibility screening, keeping in mind, that smut resistant varieties should also be treated and monitored even though the appearance of new smut races was presumed to be quite rare. The use of differential hosts is a viable option for the evaluation of pathogenic variability. However, not much of information on the use of differential hosts is available in sugarcane against the smut pathogen. Gillaspie et al., (1983) used seven sugarcane clones (Saccharum interspecific hybrids) for inoculation with S. scitamineum isolates collected from Argentina, Florida, Hawaii, Taiwan, and Zimbabwe. Six different isolates (races) could be differentiated on five of the clones under greenhouse conditions and it was concluded that this method is a valid, rapid method for isolate separation when the correct differential clones are used. It was also observed that the environment effects on the teliospores might be confounded with genetic differences amongst the test isolates which might probably complicate breeding for smut resistance. Smut pathogen being biotrophic, the inoculuma henceforth was to be maintained in the standing cane as teliospores. Slow growing fluffy white mycelia was observed from actively growing meristem tips cultured under aseptic conditions, which was further used for molecular characterization of pathogen variability. Smut isolate collection is made from different representative sugarcane growing areas in India and the pathogen

13 variability is being investigated using differential hosts and molecular markers viz. RAPD, SSR etc (Ramesh Sundar et al., 2002 - personal communication). The 20th century saw the steady spread of sugarcane smut to almost all sugar industries of the world reviewed by Presley, (1978). A widely adapted, stable smut pathotype may have been involved in this spread, explaining the lack of genetic variation in isolates collected from countries outside of Asia. Pathogenic races of sugarcane smut have been observed in several countries including two races (A and B) from Hawaii (Comstock & Heinz, 1977) and three races (1, 2, 3) reported in Taiwan (Leu et al, 1976). However, Ferreira and Comstock (1989) considered the true prevalence of races to be controversial. Many claims were based on the reaction of the same cultivar in different countries, but the interpretation of these claims was confused by test-to-test variation and the use of different inoculation methods in different countries. Xu et al., (2004) studied the genetic diversity of sugarcane smut fungus representing different provinces in Mainland China applying RAPD. Dendrogram of UPGMA cluster analysis revealed that 18 isolates of the fungus were clustered into six groups according to the dissimilarity coefficient of 0.70. The results of cluster analysis suggested that the molecular variation and differentiation could be associated with geographical origin to some extent, but not applicable to all isolates. It might be due to the frequent exchange of sugarcane varieties and clones in the recent years. Molecular diversity analysis observed no relationship between pathogen variability and host origin. Singh et al., (2005) estimated interspecies diversity within Ustilago scitaminea isolates from South Africa (SA), Reunion Island, Hawaii and Guadeloupe using RAPDs, bE mating-type gene detection, DNA sequence analysis, and spore morphological studies. Mycelial DNA of the South African isolate 14 shared 100% sequence identity with that of mycelial DNA cultured from in vitro produced teliospores of the parent cultivar. Overall, the ITS1 and ITS2 regions were found to have 96.1% and 96.9% sequence identity with a total of 17 and 21 base changes, respectively, amongst the isolates. The Reunion Island isolate was shown to be most distantly related by 3.6% to the other isolates, indicating a single clonal lineage. The lack of germination in teliospores from Guadeloupe might be attributed to changes in temperature and humidity during transportation. Raboin et al., (2007) investigated the genetic diversity and structure of different populations of the smut fungus worldwide using microsatellites by subjecting 77 distinct whips (sori) collected in 15 countries worldwide. Results indicated that the genetic diversity of either American or African S. scitamineum populations was found to be extremely low and all strains belonged to a single lineage. This lineage was also found in some populations of Asia, where most scitamineum genetic diversity S. was detected, suggesting that this fungal species originated from this region. The results obtained in this study thus suggested that the use of resistant cultivars to S. scitamineum might be an efficient and durable strategy to control sugarcane smut outside Asia. Comstock et al., (2007) comprehensively reviewed the status of genetic diversity in S. scitamineum and summarized in line with the results presented during the International Sugarcane Technologists workshop 2006. It was concluded, that the fungus originated in Asia and was disseminated to other continents on rare occasions. It was also indicated that, the resistance reaction of sugarcane clones tested in various countries was strongly influenced by the environment. The possibility of using Near Infra Red spectroscopy (NIR) in prediction of disease resistance rating for smut disease was investigated. The results were promising and the model provided acceptable predicted ratings for all the clones. Munkacsi et al., 15

(2007) suggested that domestication and cultivation of crop plants did not drive divergence and speciation of smut species on maize, sorghum, and sugarcane. The results obtained greatly weakened a hypothesis, that the speciation of crop pathogens is the necessary result of agricultural practices, and further, showed that these fungi diverged in natural populations of the fungus and host. Most importantly, the findings demonstrated that the domestication process very likely retained symbioses between the crops and scores of microbes, which had co- evolved in ancestral, natural populations. Fattah et al., (2009) attempted genotyping of the races of Ustilago species in Egypt using the chitinase gene primers. The study concluded that chitinase genes are the most suitable for genotyping study between sugarcane smut fungal isolates. The results obtained by differential display techniques showed that there were at least 10 different races from the Ustilago sp. In Egyptian field. Nzioki et al., (2010) attempted to identify presence of physiological races of sugarcane smut and the results suggested possible existence of smut races in Kenya. 2.3.5 Disease cycle

Sugarcane smut is disseminated via teliospores that are produced in the smut whip. These teliospores located either in the soil or on the plant, germinate in the presence of water (Waller, 1969). After germination they produce promycelium and undergo meiosis to create four haploid sporida. Sugarcane smut is bipolar and therefore produces two different mating types of sporida. For infection to occur, two sporida from different mating types must come together and form a dikaryon. This dikaryon then produces hyphae that penetrate the bud scales of the sugarcane plant and infect the meristematic tissue. The fungus grows within the meristematic tissue and induces formation of flowering structures which

16 it colonises to produce its Teliospores (Croft and Braithwaite, 2006). The flowering structures, usually typical grass arrows, are transformed into a whip like sorus that grows out between the leaf sheaths. At first it is covered by a thin silvery presidium (this is the host tissue) which easily peels back when desiccated to expose the sooty black-brown teliospores. These teliospores are then dispersed via wind and the cycle continues. The spores are reddish brown, round and subovoid and may be smooth to moderately echinulate. The size varies from 6.5 to 8 um. Sugarcane cultivars intended for distribution to other geographical areas should be tested for susceptibility to S. scitamineum populations in each area (Que et al., 2012). 2.3.6 Dissemination of spores

Sugarcane smut is spread by microscopic spores. The spores are particularly adapted to aerial dispersal and can be spread over great distances by wind currents (Ferreira and Comstock 1989). The whip serves as a source of spores. It has been shown that approximately one billion spores per whip per day can be released into the air. Standing sugarcane plants become infected in the buds. Since many infected buds remain dormant until sugarcane stalks are cut for seed (stalks cuttings) and planted, the use of infected seed cane is another important way that the disease is spread. Strict quarantine measures are necessary in affected areas.

Windborne spores may settle on the soil of cropped or newly prepared fields. Disease-free seed pieces may become infected if planted in soil containing viable spores. The spores, however, only survive for a short time in the soil under normal soil moisture regimes. Several species of insects have been consistently associated with smut whips and spores have been found on their bodies. These observations suggest insects 17 could play a role in spore dissemination. Although sugarcane smut has been reported on a few other members of the grass family, there are probably no important naturally occurring alternative hosts outside the Saccharum species.

2.3.7 Environment

Sugarcane smut is a very widespread disease and is prevalent in Central and South America, Africa, and South-Western Asia. Sugarcane smut has been reported in all countries that lie between 20 degrees north and south of the equator (Marin et al., 1961). The pathogen does well in hot dry weather (Riley and Jubb et al., 1999). for most of the disease cycle but requires wet conditions for teliospores to germinate.

2.3.8 Economic Impact

It is difficult to make precise assessment of the economic importance of S .scitamineum since most estimates of yield losses are based on observation and experience rather than rigorous experimentation. It is certain, however, that losses may be quite sever in susceptible varieties under conditions suitable for disease development. There are reports of yield losses of 50-73%in addition to cane yield losses. S. scitaminea also appears to reduce cane quality. Decrease in both sugar extractability and recovery, as estimated by reductions in juice purity, have been reported. S. scitaminea is also known to cause decrease in the number of millable stalks as well as in stalk diameter. In Hawaii, highly susceptible varieties showed cane yield losses of 10-15% in severely infected commercial ratoon field, while losses in sugar processing were an additional 5-7 %.( Ferreira and Comstock,.1989) Descriptions of S. scitaminea epidemics in various countries suggest that severe disease losses are associated with hot dry climates where crops

18 may experience water stress. Additionally, crop age and growth stage at the time of infection becomes more severe at the number of ratoons increases. S. scitaminea does not always pose a serious problem where it occurs. One unexplained aspect of the disease is that both incidence and severity appear to be cyclical. Severe epidemics are often followed by periods when smut can be difficult to find. To date there has been no reasonable explanation of this behavior. 2.3.9 Prevention and Control

Growing of resistant sugarcane cultivars is the best approach to smut control and has been used successfully in all sugarcane growing areas worldwide. There is a strong genetic basis for resistance. Resistant varieties have been readily available and used to control outbreaks of smut in several countries.

Rouging diseased stools has been successful in some instances usually in foreign countries where the lower wages allow repeated rouging. However, it is not practical for severe outbreaks involving commercial acreage. Rouging may be effective in seed nurseries where smut incidence is generally low.

Using disease-free seed cane is also very important for disease control. Care should be taken because this disease can be latent and show up only after planting. Disease-free planting material can usually be obtained by subjecting seed to a hot water treatment.

Various hot water treatments have been reported to be effective in controlling the smut pathogen residing in the planting setts, but it may not be practical on large scale cultivation and its effectiveness may be subject to varietal differences. The loss of bud germination due to in appropriate temperature settings needs to be handled properly (Srinivasan & Rao, 19

1968). Hardening of setts prior to hot water treatment was observed to considerably improve upon the germination of the buds. The efficacy of moist hot air treatments have been reported by Misra et al., (1978). Gupta et al., (1978) reported production of thicker and heavier canes with an increased number of millable canes due to hot water treatment. Joyce et al., (2008) attempted to utilize smut resistant varieties in genetic modification research programs leading to commercial GM crop development in Australia. Protocol optimization was done for selecting an efficient tissue culture medium to produce embryogenic celli with high transformation efficiency.

CHAPTER THREE

MATERIALS AND METHODS

3.1. Location of study:

This study was conducted in the Sugarcane Research Center, Guneid; located approximately at latitude 15°N, longitude 33°E in season 2014/2015. The soils in the experimental site are of the typical heavy clay vertisols with about 64% clay, 0.09%N, 2-8ppm available P and alkaline in reaction with pH of 8.2. Mean annual rainfall is about 112 mm falling mainly in July and August.

3.2. Test varieties

The experiments conducted included 27 newly introduced sugarcane varieties obtained from bulking plots of varieties at Gunied Sugarcane Research Station that passed quarantine periods in addition to variety Co 6806 which used as highly resistant check.

3.3. Collection and maintenance of S. scitamineum inoculum

Typical sugarcane smut whips or sori were collected from Sennar, Assalaya, Newhalfa, Gunied commercial sugarcane fields. Whips were then shade dried for 72 hrs. Thereafter, smut teliospores were extracted using a 200 mm diameter (500, 250 and a final 106) micro aperture or mesh ) laboratory sieves mounted on an Endecott’s sieve shaker model EFL 2000. the teliospores were then maintained in sealed polythene bags in the laboratory prior to use for artificial inoculation trials.

3.4. Seedbed preparation and planting materials

For all field experiments , the land was prepared according to the standard practice; by a disc plough then harrowed , leveled and ridged . The spacing between ridges was 1.5 m. plot size was I or 2 rows of 5 or 10 meter length .

3.5. Preparation of sugarcane differentials:

Three-node cuttings were prepared from each of the twenty-seven introduced varieties and commercial cultivar Co 6806. All varieties were 8-10 month old. The tested varieties were: TCP 93-4241, FG 066700, CP 99-1894, FG 03425, DP 71060, FG 03318, DB 66113, FG 03487, FG 03418, FG 03520, FG 04463, FG03372, VMC 95173, DB 70047, FG 03204, FG 06729, FG 04754, PSR 97092, BSR 97051, BJ85-34, BBZ 951034, B 89640, BJ 82118, B 93775, B 93712, B 04996, B 041291. The secured cuttings from each variety were given a long hot water treatment (HWT) at 50⁰C for 2 hr before being artificially inoculated by two methods; namely, (a) the dip methods (DM) and (b) natural spreader row method (NSRM) as described by Marchelo et al., (2008).

3.6. Inoculation protocol/ methods and field design:

All test materials were inoculated by each of two methods

3.6.1. Dip methods (DM):

The seed setts inoculated by dipping or immersion into a smut spore suspension at a concentration of 1 g smut teliospores/L of water for 15-20 minutes. The inoculated seed setts were then kept under humid condition in polythene bags for 24 hr prior to planting in the field. The plot size was 1 furrow of 10 m length and furrows spaced 1.5 m apart. A 20 cane setts were planted per plot and the plots were arranged in a randomized 22 complete block design with three replications. Sixty buds planted in each plot.

3.6.2 Natural spreading method (NSM):

In this protocol, healthy setts of the tested materials were inter-planted with susceptible cultivar NCO 376 as inoculums spreader. Spreader row material was prepared by dipping method as mentioned in 3.6.1., and planted in one speeder row (furrow) between two tested genotypes. The plot size was 1 furrow of 5 m length and furrows spaced 1.5 m apart. A 20 cane setts were planted per plot (Sixty buds in each plot) and the plots were arranged in a randomized complete block design with three replications.

3.7. Disease incidence and assessment of resistance

Disease incidence was determined from the proportion of diseased stools expressed as percentage of the total number of the stools in each plot. Resistance as expressed by reaction types was evaluated with a numerical rating scale of 1-9 where, 1=highly resistant and 9=highly susceptible as described by Satya Vir Beniwal (1978) table(1). The cumulative number of whips as an infection index was also recorded. The final genotypic reaction types in all trails were determined at the age of 16 months for plant cane (PC) and ratoons cane (RC).

3.8. Data collection

Data on (i) infection index such as (a) smut incidence on stools basis (SI%) and (b)cumulative number of smut whips (CNSW) during the growing season was determined; and (ii) epidemiological parameters namely,(a)the latent infection period (= time period from inoculation to disease symptom expression ) in days (LIP/D); (b)sustained disease

23 duration (=time from inoculation to disease symptom expression)or LIP/D to harvest in days (SDD/D).

3.9. Statistical analysis

The collected data in both methods were subjected to an analysis of variance by either the statistical software MSTAT-C or SAS and Duncan’s Multiple Ranges Test was used to locate differences between the treatment means.

3.10. Evaluation of sugarcane germplasm against smut:

Table 1: Numerical rating system for sugarcane smut according to (Satya Vir Beniwal, 1978)*

Percentage infection Rating Definition /Reaction type 0 to3% 1 Highly Resistant (HR) 4 to6% 2 Resistant (R) 7 to 9% 3 Resistant (R) 10 to12% 4 Resistant (R) 13 to25 % 5 Moderately susceptible (MS) 26 to 35% 6 Susceptible (S) 36 to 50% 7 Highly susceptible (HS) 51 to 65% 8 Highly susceptible (HS) 66 to 100% 9 Highly susceptible (HS)

CHAPTER FOUR

RESULTS

4.1 Reaction of varieties inoculated with natural infection method

Table, 2, figure, 1 and figure 2, showed that the reaction of test varieties to smut disease of sugarcane inoculated with natural infection method.

The results obtained showed that the percentage of stool infection ranged from 0% in variety CP 99-1894 that rated as highly resistant (HR) and

76% in variety BSR 97051 which rated as highly susceptible (HS). Out of

27 varieties tested, five varieties reacted as highly resistant in plant cane, namely, (CP 99-1894, FG 03204, B 89640, DB 66113, DP 71060 and one variety FG 04754 rated 2R compared to check resistant cultivar Co 6806 which was rated HR. five varieties maintained similar reactions to smut in first ratoon. The remaining varieties which reacted and rated less than resistant (R), their reaction ranged from moderately to highly susceptible according to Satya Vir (1978) scale of resistance. The smut infection in these varieties was higher in first ratoon than in plant cane. However, other than the varieties which rated as HR or R, the percentages of stools infection was observed to be higher in first ratoon (R1) than plant cane.

Table 2: Rating varieties of smut disease reactions to smut inoculated with natural infection method in plant cane (PC) and in first ratoon (R1).

Natural infection methods Natural infection methods (P.C) Ratoon, 1 cane(p.c) Cultivars Rating Rating

CP 99-1894 1HR 1HR FG 03204 1HR 1HR B 89640 1HR 1HR DB 66113 1HR 1HR DP 71060 1HR 1HR CO 6806 1HR 1HR FG 04754 2R 2R BJ 82118 2R 6S FG 03418 4R 7HS FG 03425 4R 7HS BBZ 951034 4R 6S VMC 95173 5MS 9HS FG 03372 5MS 8HS DB 70047 5MS 8HS B 93712 5MS 5MS FG 04463 5MS 6S B 93775 5MS 5MS FG 066700 6S 7HS FG 03318 6S 7HS FG 03520 6S 8HS TCP 93-4214 6S 7HS FG 03487 6S 9HS FG 06729 6S 9HS PSR97092 7HS 9HS BJ 85-34 7HS 9HS B 041291 8HS 9HS B 04996 9HS 9HS BSR 97051 9HS 9HS SE 0.90 0.91 CV% 34.81% 26.83% LSD 2.57 2.58

Figure 1: Percentage of smut stool infection in plant cane (PC) of sugarcane varieties inoculated with natural infection method

20 % stool infection stool % 10

Varieties

Figure 2: Percentage of smut stool infection in first ratoon (R1) of sugarcane varieties inoculated with natural infection methods

120

100

60 infection infection

40 stool % 20

Varieties

4.2 Reaction of varieties to smut inoculated with dip inoculation method

The reaction of test varieties to smut disease of sugarcane inoculated with dip inoculation method was presented in Table, 3, figure 3 and 4. The data revealed that the percentage of stool infection ranged from 1% in variety CP 99-1894 that rated as highly resistant (HR) and 87% in variety VMC 95173which are rated as highly susceptible (HS). The reactions of the varieties inoculated with dip method are more or less similar to that of natural method in plant cane (Table, 4 and fig. 5). Moreover, there is a positive correlation (0.56) in the results of the two methods. The remaining varieties which reacted and rated less than resistant (R), their reaction ranged from moderately to highly susceptible according to Satya Vir (1978) scale of resistance. The smut infection in these varieties was higher in first ratoon than in plant cane. However, other than the varieties which rated as HR or R, the percentages of stools infection was observed to be higher in first ratoon (R1) than plant cane.

Table 3: Rating varieties of smut disease reactions to smut inoculated with dipping infection method in plant cane PC and in first ratoon R1

Dipping infection methods plant cane(P.C) Ratoon cane (R1) Varieties Rating rating CP 99-1894 1HR 1HR CO 8606 2R 2R B 93775 4R 6S FG 04754 5R 5R DP 71060 5MS 7HS FG 066700 5MS 8HS BBZ 951034 5MS 7HS BJ 82118 5MS 5MS B 89640 6S 7HS FG 03425 6S 7HS DB 66113 6S 6S B 93712 6S 7HS B 04996 6S 8HS BJ 85-34 6S 9HS FG 03204 6S 6S TCP 93-4214 7HS 8HS FG 04463 7HS 8HS FG 03418 7HS 7HS FG 06729 8HS 9HS DB 70047 8HS 8HS FG 03372 8HS 8HS B 041291 8HS 9HS FG 03318 8HS 8HS FG 03520 8HS 8HS PSR97092 8HS 9HS BSR 97051 8HS 9HS FG 03487 9HS 9HS VMC 95173 9HS 9HS SE 0.74 0.72 CV% 21.69% 19.40% LSD(0.05) 2.09 2.03

Figure 3: Percentage of smut stool infection in plant cane (PC) of sugarcane varieties inoculated with dip infection methods.

100 90 80 70 60 50 40

% stoool % infection stoool 30 20 10 0

Varieties

Figure 4: Percentage of smut stool infection in fist ratoon (R1) of sugarcane varieties inoculated with dip infection methods

100 90 80 70 60 50 40 30

%stool %stool infection 20 10 0

Varieties

Table 4: Percentage of stool infection showed the correlation between the natural infection methods and dipping infection methods in plant cane.

Cultivars %stool infection in natural (P.C) % stool infection in dipping (P.C) CP 99-1894 0.71 0.71 FG 03204 1.55 5.69 B 89640 2.39 5.18 DB 66113 2.82 5.26 DP 71060 2.9 4.2 CO 6806 2.9 2.1 FG 04754 3.21 3.36 BJ 82118 3.21 4.94 FG 03418 3.24 6.67 FG 03425 3.36 5.2 BBZ 951034 3.67 4.38 VMC 95173 4 9.38 FG 03372 4.26 7.56 DB 70047 4.35 7.5 B 93712 4.48 5.29 FG 04463 4.53 6.49 B 93775 4.54 3.2 FG 066700 5.14 4.27 FG 03318 5.15 7.75 FG 03520 5.22 7.96 TCP 93-4214 5.29 6.15 FG 03487 5.75 9.27 FG 06729 5.8 7.09 PSR97092 6.37 8.04 BJ 85-34 6.76 5.68 B 041291 7.36 7.73 B 04996 7.8 5.34 BSR 97051 7.82 8.45 Correlation 0.56

Figure 5: Percentage of smut stool infection in plant cane (PC) of sugarcane varieties inoculated with natural infection methods )

80 70 60 50 40 30 20 10 0 B 89640 B 93712 B 93775 B 04996 B BJ 85-34 BJ CO 6806 CO BJ 82118 BJ B 041291 B FG FG 03204 FG 04754 FG 03418 FG 03425 FG 03372 FG 04463 FG 03318 FG 03520 FG 03487 FG 06729 DP 71060 DP DB 66113 DB 70047 PSR97092 BSR BSR 97051 FG FG 066700 %stool infection inatural methods (P.C methods inatural %stoolinfection CP 99-1894 CP VMC 95173 VMC BBZ 951034 BBZ TCP 93-4214 TCP

Varieties

Figure 6: Percentage of smut stool infection in plant cane (PC) of sugarcane varieties inoculated with dip infection methods

100 90 80 70 60 50 40 30 20 10 0 %stoool infection in dipping methods (P.C) methods dippingin infection %stoool B 93775 B 89640 B 93712 B 04996 B BJ 85-34 BJ CO 8606 CO BJ 82118 BJ B 041291 B FG FG 04754 FG 03425 FG 03204 FG 04463 FG 03418 FG 06729 FG 03372 FG 03318 FG 03520 FG 03487 DP 71060 DP DB 66113 DB 70047 DB PSR97092 BSR 97051 BSR FG 066700 FG CP 99-1894 CP VMC 95173 VMC BBZ 951034 BBZ Varieties 93-4214 TCP

CHAPTER FIVE

DISCUSSION

The stem or culmicolous smut (Sporisorium scitamineum) of sugarcane is cosmopolitan in distribution, and at one time or another has been important in nearly every sugarcane producing country of the world (Ferreira et al., 1980 and Peter, 1998). Management of the disease depends mainly on resistance of cane variety to the pathogen where in absence of resistant varieties losses up to 100% is expected (Croft et al., 2000). In the Sudan smut disease draws greatest attention because the disease is prevalent on all sugar estates and at times it seriously threatened the industry resulting in that two of the highest yielding varieties for both cane tonnage and sugar (Nco 310 and Nco 376) have been phased out of commercial cultivation (Nasr and Ahmed, 1974). Since then the search for an effective control measures was emphasised.

Obviousyl, the use of resistant varieties is one of the best approach to control smut disease. Infact, the aspect of evaluation of varietal disease reaction has been adopted a number of recently introduced varieteis to the Sudan and locally bred ones (Marchelo et al., 2008). In fact, reflection of the negative relation between the distribution pattern of the fungus and resistance to the disease is an important aspect in the host-pathogen interaction as it suggests the existance of a form of resistance that was different and independant of factors governing bud infection. The observation could also account for variation in incubation period for whip development.

Accordingly, this study was under taken to screen 27 newly introduced varieties of sugarcane for resistance to smut disease using two methods of

35 inoculation, dip infection and natural infection. The results revealed that out the 27 varieties tested, five ones reacted as highly resistant, |Namely, (CP 99-1894, FG 03204, B 89640, DB 66113, DP 71060 and one variety FG 04754 rated 2R compared to check resistant cultivar Co 6806 which was rated HR according to the scale Satya (1978). The categorization of sugar cane based on their reactions to inoculation with smut disease using dip and natural infection methods was also used by Nasr and Ahmed (1974); Croft et al., (2000) and Marchelo et al., (2008). The remaining varieties which reacted and were rated less than (R) are not suitable for commercial cultivation.

The results revealed that the test varieties expressed slightly higher percentage of stool infection in first ratoon than plant cane in the two methods of inoculation. These results were in line with Nasr and Ahmed (1974) and Marchelo et al., (2008) who reported the buildup of smut in successive ratoons of cane especially in susceptible varieties.

Obviously, the dip inoculation is the most widely used method for screening varieties in sugarcane breeding programs (Lee-Lovick, 1978; Ferreira et al., 1980). In this study, the results obtained from natural infection trials indicated that the dip inoculation method showed a good correlation with natural infection method. This result is in line with Ferreira et al. (1980) who reported a good correlation between the two methods in Hawaii. They reported two main constraints for the natural infection trial, compared with dip inoculation. This is because natural infection requires (i) at least two ratoon cycles to get a reliable result (Ferreira et al., 1980) and (ii) more than double the area for trial establishment an observation that encountered in this study.

Conclusions and Recommendation

Conclusions

Based on the results of this study it can be concluded that:-

 The reflection of the negative relation between the distribution pattern of the fungus and resistance to the disease is an important aspect in the host-pathogen interaction as it suggest the existance of a form of resistance that was different and independant of factors governing bud infection.  Out of 27 varieties tested using dip and natural inoculation methods, five ones were proven to be as highly resistant to smut disease, namely, (CP 99-1894, FG 03204, B 89640, DB 66113, DP 71060 and one variety FG 04754 rated 2R compared to check resistant cultivar Co 6806 which was rated HR  Variety CP 99-1894 demonstrated a very high degree of resistant to smut disease in plant cane and first ratoon in the two methods of inoculation.  The experiment obsreved that the level of smut infection in susceptible varieties was observed to be higher in first ratoon than plant cane and hence should be excluded them from commercial planting  The study revealed that the dip inoculation method is suitable for screening sugarcane varieties for resistance to smut disease as it was positively correlated with natural infection method which requires more time and space.

Recommendation

 To continue research pertaining to disease expression and rates of disease increase in ratoon crops.  More research was needed to choose whether to use percentage of stool infection or whip percentage as parameter for evaluating the level of resistance in sugarcane.  To make use of the variety CP99-1894 this expressed high level of resistance in commercial and breeding programmes.

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Appendices

Appendices (1): infection (natural p.c)

Grand Mean = 4.514 Grand Sum = 379.200 Total Count = 84

T A B L E O F M E A N S

1 2 3 Total ------1 * 4.529 126.800 2 * 4.860 136.080 3 * 4.154 116.320 ------* 1 5.290 15.870 * 2 5.140 15.420 * 3 0.710 2.130 * 4 3.363 10.090 * 5 2.907 8.720 * 6 5.153 15.460 * 7 2.827 8.480 * 8 5.750 17.250 * 9 3.240 9.720 * 10 5.220 15.660 * 11 4.530 13.590 * 12 4.260 12.780 * 13 4.000 12.000 * 14 4.353 13.060 * 15 1.553 4.660 * 16 5.800 17.400 * 17 4.000 12.000 * 18 6.377 19.130 * 19 7.820 23.460 * 20 6.760 20.280 * 21 3.670 11.010 * 22 2.397 7.190 * 23 3.213 9.640 * 24 4.547 13.640 * 25 4.487 13.460 * 26 7.800 23.400 * 27 7.367 22.100 * 28 3.867 11.600 ------

Appendices (2):

A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 6.981 3.491 1.4138 0.2521 2 Factor A 27 249.008 9.223 3.7356 0.0000 -3 Error 54 133.316 2.469 ------Total 83 389.305 ------

Appendices (3):

Infection (dipping p.c)

Grand Mean = 5.911 Grand Sum = 496.550 Total Count = 84

T A B L E O F M E A N S

1 2 5 Total ------1 * 6.726 188.320 2 * 5.668 158.710 3 * 5.340 149.520 ------* 1 6.150 18.450 * 2 4.270 12.810 * 3 1.273 3.820 * 4 5.207 15.620 * 5 4.200 12.600 * 6 7.753 23.260 * 7 5.267 15.800 * 8 9.277 27.830 * 9 6.673 20.020 * 10 7.967 23.900 * 11 6.493 19.480 * 12 7.560 22.680 * 13 9.383 28.150 * 14 7.507 22.520 * 15 5.697 17.090 * 16 7.097 21.290 * 17 3.367 10.100 * 18 8.043 24.130 * 19 8.457 25.370 * 20 5.680 17.040 * 21 4.387 13.160 * 22 5.183 15.550 * 23 4.947 14.840 * 24 3.203 9.610 * 25 5.297 15.890 * 26 5.347 16.040 * 27 7.733 23.200 * 28 2.100 6.300 ------

Appendices (4):

A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 29.365 14.682 8.9320 0.0004 2 Factor A 27 341.844 12.661 7.7022 0.0000 -3 Error 54 88.765 1.644 ------Total 83 459.974 ------

Appendices (5):

Natural infection methods (R1)

Grand Mean = 6.341 Grand Sum = 532.672 Total Count = 84

T A B L E O F M E A N S

1 2 3 Total ------1 * 6.815 190.810 2 * 6.332 177.302 3 * 5.877 164.560 ------* 1 7.623 22.870 * 2 6.517 19.550 * 3 0.710 2.130 * 4 6.727 20.180 * 5 6.627 19.880 * 6 7.327 21.982 * 7 5.423 16.270 * 8 7.963 23.890 * 9 6.673 20.020 * 10 7.787 23.360 * 11 7.773 23.320 * 12 7.133 21.400 * 13 9.097 27.290 * 14 6.940 20.820 * 15 5.547 16.640 * 16 7.860 23.580 * 17 2.270 6.810 * 18 7.753 23.260 * 19 8.130 24.390 * 20 8.167 24.500 * 21 5.963 17.890 * 22 6.757 20.270 * 23 4.780 14.340 * 24 4.467 13.400 * 25 4.830 14.490 * 26 6.160 18.480 * 27 9.000 27.000 * 28 1.553 4.660 ------

Appendices (6):

A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 12.308 6.154 4.0681 0.0226 2 Factor A 27 352.517 13.056 8.6307 0.0000 -3 Error 54 81.689 1.513 ------Total 83 446.514 ------

Appendices (7):

Infection dipping methods (R1)

Grand Mean = 5.891 Grand Sum = 494.820 Total Count = 84

T A B L E O F M E A N S

1 2 4 Total ------1 * 6.197 173.510 2 * 5.492 153.790 3 * 5.983 167.520 ------* 1 7.223 21.670 * 2 7.040 21.120 * 3 0.710 2.130 * 4 6.597 19.790 * 5 1.610 4.830 * 6 6.653 19.960 * 7 2.453 7.360 * 8 7.763 23.290 * 9 6.330 18.990 * 10 7.403 22.210 * 11 4.960 14.880 * 12 6.490 19.470 * 13 8.940 26.820 * 14 8.223 24.670 * 15 1.553 4.660 * 16 7.970 23.910 * 17 2.063 6.190 * 18 9.007 27.020 * 19 8.880 26.640 * 20 9.267 27.800 * 21 6.000 18.000 * 22 2.827 8.480 * 23 5.347 16.040 * 24 3.257 9.770 * 25 4.487 13.460 * 26 7.307 21.920 * 27 10.033 30.100 * 28 4.547 13.640 ------

Appendices (8):

A N A L Y S I S O F V A R I A N C E T A B L E

K Degrees of Sum of Mean F Value Source Freedom Squares Square Value Prob ------1 Replication 2 7.301 3.650 1.4612 0.2410 2 Factor A 27 571.544 21.168 8.4735 0.0000 -3 Error 54 134.901 2.498 ------Total 83 713.746